The Role Of TLR4 In Anti-β2GPI Antibody Induced Adhesion Molecules And Tissue Factor Expression In Mouse Artery

Objective:The main clinical features of APS (antiphospholipid syndrome) are high titers of antiphospholipid antibodies (aPLs) in serum, recurrent thrombosis in vein and artery, thrombocytopeni, and recurrent abortion. Thrombosis is the leading cause of death in patients with APS, high titer aPLs is thought to play a key role in APS associated thrombosis. Negative phospholipid binding protein β2-glycoprotein I (β2-glycoprotein I, β2GPI) is a key target of anti-β2GPI Ab antigen.Many in vitro studies have shown that anti-β2GPI Ab can induce increased expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and tissue factor (TF) in ECs through TLR4/NF-κB pathway. However, the studies on mechanism of anti-β2GPI Ab induced ECs dysfunction in vivo are still rare. Basing on this background, anti-β2GPI Ab was used in the stimulation of C3H/HeN mice (TLR4 intact) and C3H/HeJ mice (TLR4 defective) to investigate whether TLR4 itself or its downstream signal molecule participate in ECs activation and expression of adhesion molecules and TF induced by anti-β2GPI Ab.Methods:(1) C3H/HeN mice and C3H/HeJ mice were intraperitoneal injected with anti-β2GPI Ab (experiment group), LPS (positive control group) or saline (negative control group) at Oh and 48th. The protein expression of VCAM-1, ICAM-1 and E-selectin in endarterium artery was detected by immunohistochemistry at 72th to show the effect of TLR4 in anti-β2GPI Ab induced TLR4 activation.(2) Western blot and RT-qPCR (Real-time quantitative PCR) were employed to measure protein and mRNA expression of ICAM-1, VCAM-1 and E-selectin in artery homogenate of C3H/HeN mice and C3H/HeN mice after anti-β2GPI Ab, LPS or saline stimulation.(3)In order to evaluate the effect of TLR4 on expression and activation of TF in ECs, C3H/HeN mice and C3H/HeJ mice were stimulated with anti-β2GPI Ab, then RT-qPCR and TF activity kit were used to measure the expression of TF mRNA in artery homogenate.(4)In order to study the role of TLR4/NF-κB pathway in anti-β2GPI Ab induced ECs activation, the level of total protein and phosphorylated protein of p38MAPK and NF-κB p65 aorta homogenate of C3H/HeN mice and C3H/HeJ mice were measured by western blot.Results:(1)The positive dyeing rate of VCAM-1, ICAM-1, E-selectin molecules in artery endothelial cell layer of C3H/HeN mice were increased after injection of anti-β2GPI Ab or LPS, but not saline. Furthermore, no positive expression of these molecules can be tested in C3H/HeJ mice after anti-β2GPI Ab stimulation. The difference between C3H/HeN mice and C3H/HeJ mice has significant difference.(2) After anti-β2GPI Ab stimulation, the mRNA and protein expression of VCAM-1、ICAM-1 and E-selectin in C3H/HeN mice aorta homogenate are increased compared with C3H/HeJ mice, the difference have statistical significance (p<0.05)(3) The mRNA level and activity of TF in artery homogenate of C3H/HeN mice were elevated compared with that of C3H/HeJ mice, the difference have statistical significance.(4) After anti-β2GPI Ab stimulation, the phosphorylated protein level of p38MAPK and NF-kB p65 in artery homogenate of C3H/HeN mice were elevated compared with that of C3H/HeJ mice, the difference have statistical significance.Conclusions:(1)AntiβP2GPI Ab can induce the activation of vascular ECs and the elevated expression of adhesion molecules as well as TF, this is similar to the results of former in vitro experiments.(2) The lack of TLR4 gene can obviously but partly suppress ECs activation induced by anti-β2GPI Ab, this result implies the activation of ECs induced by anti-β2GPI Ab are partly depend on the effect of TLR4.(3) The high expression and phosphorylation of TLR4 downstream signal molecules, p38MAPK and NF-kB after anti-β2GPI Ab stimulation implied the involvement of TLR4/NF-κB signal pathway in anti-β2GPI Ab induced ECs activation in mice vascular ECs.